Foxp3+ regulatory T cels (Tregs) are critical for the regulation of immune homeostasis, organ-specific tolerance, and inflammation. Because of the central role of Tregs in immune modulation, and the prevalence of Tregs in various human cancers, many emerging therapeutic strategies have focused on the modulation or depletion of Tregs concomitant with vaccine administration, in an effort to stimulate effective anti-tumor immune responses. However, little is known about the developmental origins, specificity, and in situ function of tumor-infiltrating Tregs. To date, studies of Foxp3+ Tregs reactive to tumor-associated antigens have relied on the ectopic expression of model foreign antigens on tumor cells. However, since the natural antigens recognized by Tregs are unknown, these experimental systems may not correctly recapitulate several fundamental aspects of Treg biology. Therefore, in order to understand the true nature of tumor-associated Tregs, it is paramount to develop novel systems by which to directly analyze endogenous, antigen-specific Treg populations. In our ongoing work, we have identified a population of naturally occurring Foxp3+ Tregs expressing a conserved TCR?? that is highly enriched by antigen-driven selection in mice with prostate cancer (hereafter, cells of this specificity will be referred to as RT3 T cells), and generated transgenic mice expressing this RT3 Treg TCR. The overall objective of this proposal is to understand the development, lineage plasticity, and specificity of the endogenous RT3 Treg response. It is our central hypothesis that RT3 T cells are natural Foxp3+ Tregs that develop in the thymus, and that these Tregs are responsive to shared antigens and common stimuli that are not specific to prostate cancer. To address this hypothesis, we will use complementary approaches: a) analysis of the development, differentiation, and stability of purified TCR transgenic cell populations following transfer into tumor-bearing hosts, and b) quantification of canonical RT3 TCR? transcripts in endogenous T cell subsets from tumor-bearing mice using deep TCR sequencing. We plan to test our central hypothesis and accomplish the objectives of this application by pursuing the following three specific aims. In Aim 1, we will test the hypothesis that Foxp3neg effector T cells and Foxp3+ Tregs of the same RT3 specificity develop concurrently in tumor-bearing mice, as a result of Foxp3+ Treg differentiation into Foxp3neg cells. In Aim 2, we will test the hypothesis that Foxp3+ RT3 Tregs are a population of natural Tregs that develop in the thymus, and are highly enriched in prostate tumors by antigen-driven selection. Finally, in Aim 3, we will test the hypothesis that RT3 Tregs are not prostate cancer-specific, but are instead responsive to shared antigens and common stimuli. Completion of these aims will provide fundamental insight into the biology of regulatory T cells, which will guide the development of therapeutic strategies aimed at the induction of cancer regression through the selective modulation or depletion of tumor-associated Tregs.